Methods of treating myointimal proliferation

ABSTRACT

The present invention provides a method of treating myointimal proliferation by administering a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/309,047, filed on Dec. 11, 2018, which is a 35 U.S.C. 371 national stage filing of International Application No. PCT/US2017/037695, filed on Jun. 15, 2017, which claims the benefit of U.S. Provisional Application No. 62/350,936, filed on Jun. 16, 2016. The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 5, 2022, is named INZN_001_C01US_SeqListing.txt and is 89370 bytes in size.

BACKGROUND

Myointimal proliferation (also known as myointimal hyperplasia) is an arterial wall smooth muscle cell (SMC) proliferative disorder (Painter, TA, Artif Organs. 1991 February; 15(1):42-55). Specifically, myointimal proliferation involves the migration and proliferation of vascular smooth muscle cells (VSMCs), as well as the involvement of the extracellular matrix in the intima i.e., the innermost coat of a blood vessel consisting of an endothelial layer backed by connective tissue and elastic tissue (see, e.g., Kraiss L W, Clowes A W, In: Sumpio S A N, ed. The Basic Science of Vascular Disease. New York, N.Y.: Futura Publishing; 1997:289-317; and Yang Z, Luscher T F. Eur Heart J. 1993; 14(suppl):193-197).

Ectonucleotide pyrophosphatase pyrophosphorylase 1 (NPP1) is an ectoenzyme that cleaves ATP to produce extracellular pyrophosphate (PPi). Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/ENPP1/PC-1) deficiency is a rare disease caused by mutations in NPP1, a type II transmembrane glycoprotein. NPP1 cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. NPP1 deficiency has been associated with idiopathic infantile arterial calcification (IIAC), insulin resistance, hypophosphatemic rickets, and ossification of the posterior longitudinal ligament of the spine. IIAC, a rare autosomal recessive and nearly always fatal disorder, is characterized by calcification of the internal elastic lamina of muscular arteries and stenosis due to myointimal proliferation. There are more than 160 cases of IIAC that have been reported world-wide. The symptoms of the disease most often appear by early infancy, and the disease is lethal by 6 months of age, generally because of ischemic cardiomyopathy, and other complications of obstructive arteriopathy including renal artery stenosis.

Although defects in the NPP1 protein have been implicated in such serious disease as IIAC, currently no treatment is available for those who are affected by the disease. Current therapeutic options have limited efficacy and undesirable and/or unacceptable side effects. Braddock, D. et al., (WO 2014/126965A2) discloses compositions and methods for treating pathological calcification and ossification by administering NPP1. Quinn, A. et al., (WO 2012/125182A1) discloses a NPP1 fusion protein to treat conditions including Generalized Arterial Calcification of Infancy (GACI), arterial calcification, insulin resistance, hypophasphatemic rickets, and ossificaiton of the posterior longitudinal ligament of the spine.

In spite of considerable research in the field, there is a continuing need for new therapies to effectively treat NPP1-deficiencies, including myointimal proliferation disorders

SUMMARY OF THE INVENTION

The present invention relates to uses of isolated recombinant human soluble NPP1 that lacks N-terminal cytosolic and transmembrane domains and fusion proteins thereof for the treatment of NPP1-deficiency and/or myointimal proliferation disorders. Any disorder that is characterized by myointimal proliferation is within the scope of the present invention.

In one aspect, methods for treating a human patient having detected myointimal proliferation (e.g., as assessed by immunohistochemical detection, ultrasound (e.g., intravascular ultrasonography, carotid ultrasound, or contrast-enhanced ultrasound (CEU)), X-ray computed tomography (CT), nuclear imaging (e.g., positron emission tomography (PET) or single-photon emission computed tomography (SPECT)), optical imaging, or contrast enhanced image) are provided, the method comprising administering to the patient one or more doses of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.

In another aspect, the methods of treating myointimal proliferation (e.g., as assessed by immunohistochemical detection) in a human patient, are provided, the method comprising: a) identifying a human patient as having myointimal proliferation and b) administering to the identified patient one or more doses of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.

In one embodiment, a NPP1 fusion protein is administered. Preferred fusion proteins comprise and NPP1 component an Fc region of an immunoglobulin and, optionally, a targeting moiety. In one embodiment, the targeting moiety is Asp₁₀ (SEQ ID NO: 18). In another embodiment, the targeting moiety comprises at least eight consecutive aspartic acid or glutamic acid residues (SEQ ID NOS 20 and 21, respectively). Particular NPP1 fusion proteins for administration in accordance with the methods disclosed herein have the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

In another embodiment, the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP). In another embodiment, administration of a recombinant hsNPP1 according to the methods described herein is sufficient to normalize adenosine or adenosine monophosphate (AMP) production in the human patient. In another embodiment, administration of a recombinant hsNPP1 according to the methods described herein is sufficient to prevent arterial stenosis in the patient.

Any suitable amount of the recombinant hsNPP1 can be administered to the human patient. In one embodiment, the hsNPP1 is administered in one or more doses containing about 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0 mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 mg/kg, or 20.0 mg/kg. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg NPP1. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg NPP1.

The time period between doses of the hsNPP1 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month. In one embodiment, the administration is weekly, bi-weekly, or monthly.

The recombinant hsNPP1 can be administered in any suitable way, such as intravenously, subcutaneously, or intraperitoneally.

The recombinant hsNPP1 can be administered in combination with one or more additional therapeutic agents. Exemplary therapeutic agents include, but are not limited to Bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and varfarin. In one embodiment, the recombinant hsNPP1 and additional therapeutic agent are administered separately and are administered concurrently or sequentially. In one embodiment, the recombinant hsNPP1 is administered prior to administration of the additional therapeutic agent. In another embodiment, the recombinant hsNPP1 is administered after administration of the additional therapeutic agent. In another embodiment, the recombinant hsNPP1 and additional therapeutic agent are administered together.

In one embodiment, the patient does not have arterial calcification. In one another, the patient does have arterial calcification.

In another aspect uses of an isolated recombinant human sNPP1, fragment or fusion protein thereof are provided. In one embodiment, the use of an isolated recombinant human sNPP1, fragment or fusion protein thereof for the manufacture of a medicament for treating myointimal proliferation is provided. In another embodiment, the invention provides the use of an isolated recombinant human sNPP1, fragment or fusion protein thereof for treating myointimal proliferation. In one embodiment, the myointimal proliferation is not associated with arterial calcification. In another embodiment, the myointimal proliferation is associated with arterial calcification.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict mRNA (FIG. 1A) and protein (FIG. 1B) ENPP1 expression in human primary VSMCs from six different donors, as well as enzyme activity from three of the donors (FIG. 1C).

FIGS. 2A-2B depict inhibition of ENPP1 mRNA expression relative to a negative control. Specifically, FIG. 2A depicts inhibition of ENPP1 mRNA expression relative to a negative control for five siRNA constructs, 48 hours post siRNA transfection. FIG. 2B depicts inhibition of ENPP1 mRNA expression relative to a negative control six and eleven days post siRNA transfection with construct #4.

FIGS. 3A-3B depict the effect of silencing ENPP1 by siRNA on proliferation of human primary VSMCs from two different donors.

FIGS. 4A-4B depict the effect of silencing ENPP1 by siRNA on human primary VSMC cell growth on Day 3 (FIG. 4A) and Day 4 (FIG. 4B).

FIGS. 5A-5B depict the effect of siRNA silencing on levels of ENPP1 mRNA expression (FIG. 5A), cell growth (FIG. 5B), and enzyme activity (FIG. 5C) in rat VSMCs.

FIGS. 6A-6B depict the effect of adenosine (FIG. 6A), AMP (FIG. 6A), and PPi (FIG. 6B) on proliferation of rat VSMCs that were knocked down with ENPP1.

FIG. 7 depicts the effect of bisphosphonate on proliferation of rat VSMCs.

FIGS. 8A-8B depict the effect of silencing ENPP1 using different siRNA constructs on proliferation in rat VSMCs. Specifically, FIG. 8A depicts the effect of silencing ENPP1 by two siRNA on proliferation of rat VSMCs. FIG. 8B depicts inhibition of ENPP1 mRNA expression relative to a negative control for two siRNA constructs, 48 hours post siRNA transfection.

FIG. 9A depicts the effect of Ad-mENPP1/Ad-rENPP1 on Mouse/Rat ENPP1 messenger RNA specifically in rat VSMCs. FIG. 9B shows the distribution of GFP following co-transfection of siRNA and Ad-GFP.

FIG. 10 shows that siRNA specific to rat ENPP1 partially affected Ad-mENPP1 on Mouse ENPP1 messenger RNA specifically in co-transfection siRNA with Ad-mRNPP1 in rat VSMCs.

FIG. 11 depicts the effect of Ad-rENPP1 on ENPP1 protein expression.

FIGS. 12A-12B depict the effect of mENPP1 overexpression on enzyme activity in rat VSMCs (FIG. 12A) and A10 cells (FIG. 12B).

FIGS. 13A-13B shows that silencing ENPP1 increases proliferation on rat VSMCs and overexpression of mouse or rat ENPP1 inhibits proliferation on rat VSMCs that either downregulated ENPP1 expression (si-rENPP1) or normal ENPP1 expression (si-NC).

FIG. 14 depicts the effect of silencing ENPP1 and overexpression of mouse or rat ENPP1 on rat VSMCs cell growth.

FIG. 15 shows that FBS has a negative effect on stability of ATP in cultured supernatant. CM contains 5% FBS, Starvation media contains 0.25% FBS. ATP function was reduced over 60% in CM after 30 minutes in culture at 37° C.

FIG. 16 shows that FBS has a negative effect on stability of ATP in cultured supernatant. CM contains 5% FBS, Starvation media contains 0.25% FBS. ATP function was reduced over 87% after 2 hours in culture at 37° C.

FIG. 17 shows that FBS has a negative effect on stability of ATP in cultured supernatant. CM contains 5% FBS, Starvation media contains 0.25% FBS. ATP function was almost entirely lost after 24 hours in culture at 37° C.

FIGS. 18A-18B shows that heat denatured human/mouse ENPP1-Fc protein completely lost their enzymatic activity.

FIGS. 19A-19C depicts the effect of murine ENPP1-Fc protein (FIG. 19A), human ENPP1-Fc (FIG. 19B) and human ENPP1-FC-D10 (FIG. 19C) on proliferation of rat primary VSMCs.

FIGS. 20A-20C depict ENPP1 expression by human primary VSMCs and human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs), as assessed by qRT-PCR (FIG. 20A) and Western Blot (FIGS. 20B-C).

FIG. 21 depicts the effect of silencing ENPP1 by siRNA on the growth of human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs).

FIGS. 22A-22C depict the effect of murine (FIG. 22A) and human ENPP1-Fc (FIGS. 22B-22C) protein on the proliferation of human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs).

FIG. 23 depicts PPi levels in the supernatant, as assessed by a PPi assay using human iVSMCs.

FIG. 24 depicts the effect of bisphosphonate on proliferation of human iVSMCs.

FIG. 25 is a diagram of the carotid artery ligation and sectioning for histological analysis. Five μm sections were cut spanning 250 μm from the point of ligation. Every fifth section was analyzed.

FIG. 26 is a histological analysis (Von Gieson's stain) of sections either 100 (upper) or 200 (lower) μm from point of ligation from wild-type (WT), ttw/ttw, vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw/mice from left to right, respectively. The internal elastic lamina (IEL), external elastic lamina (EEL) and lumen (L) are indicated by arrows. The scale bar represents 100 μm.

FIGS. 27A-27C are a morphometric quantitation of medial (FIG. 27A) and intimal areas (FIG. 27B), as well as the I/M ratio (FIG. 27C). *p<0.001.

FIGS. 28A-28B show the effect of therapeutic Enpp1 treatment on intimal hyperplasia in ttw/ttw mouse started 7 days after carotid ligation. FIG. 28A shows the degree of intimal hyperplasia 7 days post ligation in WT and ttw/ttw mice. FIG. 28B shows the histological analysis (Von Gieson's stain) of sections either 100 (upper) or 200 (lower) μm from point of ligation from WT, vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw mice from left to right, respectively. The internal elastic lamina (IEL), external elastic lamina (EEL) and lumen (L) are indicated by arrows. The scale bar represents 100 μm.

FIGS. 29A-29C are a morphometric quantitations of medial (FIG. 29A) and intimal areas (FIG. 29B), as well as the I/M ratio (FIG. 29C) on treatment day 14. *p<0.05, **p<0.01.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention, the preferred methods and materials are described.

For clarity, “NPP1” and “ENPP1” refer to the same protein and are used interchangeably herein.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, the term “fragment”, with regard to NPP1 proteins, refers to an active subsequence of the full-length NPP1. A “fragment” of a protein or peptide can be at least about 20 amino acids in length; for example, at least about 50 amino acids in length; at least about 100 amino acids in length; at least about 200 amino acids in length; at least about 300 amino acids in length; or at least about 400 amino acids in length (and any integer value in between). The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. Thus, a protein “comprising at least a portion of the amino acid sequence of SEQ ID NO: 1” encompasses the full-length NPP1 and fragments thereof.

An “isolated” or “purified” soluble NPP1 protein or biologically active fragment or fusion protein thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NPP1 protein, biologically active fragment or NPP1 fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of NPP1 protein, biologically active fragment, or NPP1 fusion protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of NPP1 protein, biologically active fragment or NPP1 fusion protein having less than about 30% (by dry weight) of non-NPP1 protein/fragment/fusion protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-NPP1 protein/fragment/fusion protein, still more preferably less than about 10% of non-NPP1 protein/fragment/fusion protein, and most preferably less than about 5% non-NPP1 protein/fragment/fusion protein. When the NPP1 protein, fusion protein, or biologically active fragment thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

As used herein, the term “subject” encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like.

As used herein, the term “therapeutically effective amount” refers to a nontoxic but sufficient amount of an agent (e.g., hsNPP1 proteins) which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

As used herein, “myointimal proliferation” (also referred to as “myointimal hyperplasia”) refers to abnormal proliferation of the smooth muscle cells of the vascular wall (e.g., the intima of a blood vessel).

The term “treating” includes the application or administration of the NPP1 proteins, active fragments and fusion proteins of the invention to a subject, or application or administration of NPP1 proteins, active fragments and fusion proteins of the invention to a subject who has myointimal proliferation, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, preventing, improving, or affecting the disease or disorder. The term “treating” refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being. Treatment may be therapeutic or prophylactic. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination.

Methods of Treatment

The present invention relates to uses of an isolated recombinant human soluble NPP1 (“sNPP1”) which lacks an N-terminal portion (i.e., lacking cytosolic and transmembrane domains) and fusion proteins thereof for the treatment of NPP1-associated diseases, such as myointimal proliferation.

The subject can be a human patient having deficiencies in NPP1 activity (NPP1 deficiency). In one embodiment, the patient exhibits low levels of pyrophosphate and/or suffers from a disease or disorder associated with low levels of pyrophosphate. In another embodiment, the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP).

Generally, the dosage of fusion protein administered to a subject will vary depending upon known factors such as age, health and weight of the recipient, type of concurrent treatment, frequency of treatment, and the like. Usually, a dosage of active ingredient (i.e., fusion protein) can be between about 0.0001 and about 50 milligrams per kilogram of body weight. Precise dosage, frequency of administration and time span of treatment can be determined by a physician skilled in the art of administration of therapeutic proteins.

A preferred embodiment of the present invention involves a method of treating myointimal proliferation, which includes the step of identifying a human patient as having myointimal proliferation and administering to the identified patient a therapeutically effective amount of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.

As defined herein, a therapeutically effective amount of protein (i.e., an effective dosage) ranges from about 0.001 to 50 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of protein can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of protein used for treatment may increase or decrease over the course of a particular treatment.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 50 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. In one embodiment, the hsNPP1 is administered in one or more doses containing about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0 mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 mg/kg, 20.0 mg/kg, 21.0 mg/kg, 22.0 mg/kg, 23.0 mg/kg, 24.0 mg/kg, 25.0 mg/kg, 26.0 mg/kg, 27.0 mg/kg, 28.0 mg/kg, 29.0 mg/kg, 30.0 mg/kg, 31.0 mg/kg, 32.0 mg/kg, 33.0 mg/kg, 34.0 mg/kg, 35.0 mg/kg, 36.0 mg/kg, 37.0 mg/kg, 38.0 mg/kg, 39.0 mg/kg, 40.0 mg/kg, 41.0 mg/kg, 42.0 mg/kg, 43.0 mg/kg, 44.0 mg/kg, or 45.0 mg/kg. In another embodiment, about 0.5 to about 30 mg, about 0.5 to about 20 mg, about 0.5 to about 10 mg, or about 0.5 to about 5 mg are administered to the patient. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg hsNPP1. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg hsNPP1. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.

Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments. In one embodiment, in the range of between about 0.1 to 20 mg/kg body weight, one time per week, twice per week, once in about 10 days, once in about 12 days, once in about 14 days, once in about 17 days, once in about 20 days, once in about about 25 days, or once in about 30 days. In one embodiment, the time period between doses of the hsNPP1 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month. In another embodiment, the therapeutically effective dose of sNPP1, biologically active fragment or fusion protein thereof is administered to a patient between one time every 5 days and one time every 30 days for a period of time determined by a practitioner of skill in the art of medical sciences. In another embodiment, the period of time will be the remainder of the patient's life span. In another embodiment, the dosing frequency is between one time every 5 days and one time every 25 days. In another embodiment, the dosing frequency is between one time every 5 days and one time every 21 days. In another embodiment, the dosing frequency is between one time every 7 days and one time every 14 days. hsNPP1, biologically active fragment or fusion protein thereof can be administered one time every 5 days, one time every 6 days, one time every 7 days, one time every 8 days, one time every 9 days, one time every 10 days, one time every 11 days, one time every 12 days, one time every 13 days, or one time every 14 days. In some embodiments, hsNPP1, biologically active fragment or fusion protein thereof is administered about weekly. In other embodiments, sNPP1, biologically active fragment or fusion protein thereof is administered about bi-weekly. In one embodiment, the dosing frequency is one time about 30 days. It will also be appreciated that the effective dosage of soluble sNPP1 protein, biologically active fragment or fusion protein thereof used for the treatment may increase or decrease over the course of a particular treatment.

In one embodiment, about 1 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 2 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 3 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 4 mg/kg of sNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 5 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 6 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 7 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 8 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 9 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 10 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week.

hsNPP1, biologically active fragment or fusion protein can be administered by, for example, subcutaneous injections, intramuscular injections, and intravenous (IV) infusions or injections.

In one embodiment, hsNPP1, biologically active fragment or fusion protein is administered intravenously by IV infusion by any useful method. In one example, hsNPP1, biologically active fragment or fusion protein can be administered by intravenous infusion through a peripheral line. In another example, hsNPP1, biologically active fragment or fusion protein can be administered by intravenous infusion through a peripherally inserted central catheter.

In another embodiment, hsNPP1, biologically active fragment or fusion protein is administered intravenously by IV injection.

In another embodiment, hsNPP1, biologically active fragment or fusion protein is administered via intraperitoneal injection.

In another embodiment, hsNPP1, biologically active fragment or fusion protein is administered by subcutaneous injections.

In another embodiment, hsNPP1, biologically active fragment or fusion protein is administered by intramuscular injections.

In still another embodiment, hsNPP1, biologically active fragment or fusion protein is administered via a pharmaceutically acceptable capsule of the therapeutic protein. For example, the capsule can be an enteric-coated gelatin capsule.

In one embodiment, the method involves administering the soluble NPP1 protein or NPP1 fusion protein of the invention alone, or in combination with other agent(s). Exemplary therapeutic agents include, but are not limited to bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and varfarin. In one embodiment, the method involves administering an NPP1 protein or an NPP1 fusion protein of the invention as therapy to compensate for reduced or aberrant NPP1 expression or activity in the subject having an NPP1-deficiency or other associated disease or disorder. In one embodiment, the isolated sNPP1 proteins, fragments, and fusion proteins can be administered before, after or concurrently with the agent or can be co-administered with other known therapies. Co-administration of the isolated sNPP1 proteins, fragments, and fusion proteins of the present invention with other therapeutic agents may provide two agents which operate via different mechanisms which yield an increased therapeutic effect. Such co-administration can solve problems due to development of resistance to drugs. In particular aspects, this disclosure relates to a method for reducing myointimal proliferation in a subject in need thereof.

The methods described herein provide a way to reduce myointimal proliferation in a subject (e.g., human patient). In one embodiment, the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP). In another embodiment, administration of a recombinant hsNPP1 according to the methods described herein is sufficient to normalize adenosine or adenosine monophosphate (AMP) production in the human patient. In another embodiment, administration of a recombinant hsNPP1 according to the methods described herein is sufficient to prevent arterial stenosis in the patient.

sNPP1

The present invention employs soluble NPP1 (e.g., hsNPP1) that has a biologically active NPP1 domain of NPP1 (i.e., NPP1 components that contain at least one extracellular catalytic domain of naturally occurring NPP1 for the pyrophosphatase and/or phosphodiesterase activity). The soluble NPP1 proteins of the invention comprise at least the NPP1 domain essential to carry out the pyrophosphatase and/or phosphodiesterase activity.

In one embodiment, the soluble NPP1, fragment, and fusion proteins thereof can form functional homodimers or monomer. In another embodiment, a soluble NPP1 protein or NPP1 fusion protein thereof can be assayed for pyrophosphatase activity as well as the ability to increase pyrophosphate levels in vivo.

Described herein are various amino acid sequences of soluble NPP1 compounds, fusion partners and fusion proteins that are suitable for use according to the methods provided herein. SEQ ID NO:5 shows the amino acid sequences of a soluble NPP1 containing amino acids from 107 to 925 of SEQ ID NO:1. SEQ ID NO:6 shows the amino acid sequence of a soluble NPP1 containing amino acids from 187 to 925 of SEQ ID NO:1. SEQ ID NO:7 shows the amino acid sequence of the Fc region of human IgG1 including the hinge region. SEQ ID NO:8 shows the amino acid sequence of the Fc of human IgG1 including a partial hinge region. SEQ ID NO:9 shows the amino acid sequence of a NPP1-Fc fusion protein. The NPP1 component contains SEQ ID NO:5, and the Fc sequence includes the hinge region. SEQ ID NO:10 shows the amino acid sequence of a NPP1-Fc fusion protein. The soluble NPP1 contains SEQ ID NO:5, and the Fc sequence includes the partial hinge region. SEQ ID NO:1 shows the amino acid sequence of a NPP1-Fc fusion protein. The soluble NPP1 contains SEQ ID NO:6, and the Fc sequence includes the hinge region. SEQ ID NO:12 shows the amino acid sequence of a NPP1-Fc fusion protein. The soluble NPP1 contains SEQ ID NO:6, and the Fc sequence includes the partial hinge region.

Preferred soluble NPP1 proteins and NPP1 fusion proteins of the invention are enzymatically active in vivo (e.g., human). In one embodiment, the soluble protein comprises amino acid sequence having at least 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to the following sequence:

(SEQ ID NO: 2) PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHTW TCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCE SINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYT KNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKE KFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGS VPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKA LQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYL GDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPY LKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDN VFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTH GSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIED FQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQD ILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKV SYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKY AEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFI VLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELL MLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED

SEQ ID NO:2 is the amino acid sequence of a sNPP1 that contains the cysteine-rich region, catalytic region and c-terminal region.

Any desired enzymatically active form of soluble NPP1 can be used in the methods described herein. The enzymatically active sNPP1 can increase PPi levels in suitable enzymatic assays, and can be assayed for pyrophosphatase activity, phosphodiesterase activity, or pyrophosphatase and phosphodiesterase activity. Typically, the sNPP1 contains at least an NPP1 component that lacks the N-terminal cytosolic and transmembrane domains of naturally occurring transmembrane NPP1.

SEQ ID NO:1 is the amino acid sequence of wild-type NPP1 protein. The cytosolic and transmembrane regions are underlined. The potential N-glycosylation sites are in bold. The amino acid motif “PSCAKE” (SEQ ID NO:17) in bold is the start of a soluble NPP1 which includes the cysteine rich region.

In preferred aspects, the NPP1 component contains the cysteine-rich region (amino acids 99-204 of SEQ ID NO:1) and the catalytic region (amino acids 205-591 of SEQ ID NO:1) of naturally occurring human NPP1. Typically, the NPP1 component also includes the C-terminal region (amino acids 592 to 925 of SEQ ID NO:1), and has the amino acid sequence of SEQ ID NO:2. However, the C-terminal region can be truncated if desired. Accordingly, preferred NPP1 components include the cysteine-rich region and catalytic region of human NPP1 (amino acids 99-591 of SEQ ID NO:1) or the cysteine-rich region, the catalytic region and the C-terminal region of human NPP1 (SEQ ID NO:2). Other preferred NPP1 components contain only a portion of the cysteine-rich domain and have the sequence of amino acids 107 to 925 of SEQ ID NO:1 or amino acids 187 to 925 of SEQ ID NO:1.

The cysteine rich region of NPP1 (i.e., amino acids 99 to 204 of SEQ ID NO: 1) can facilitate dimerization of the sNPP1. The sNPP1, including fusion proteins, can be in the form of a monomer of functional homodimer.

The amino acid sequence of the NPP1 component can be a variant of the naturally occurring NPP1 sequence, provided that the NPP1 component is enzymatically active. NPP1 variants are enzymatically active and have at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 96% amino acid sequence identity to the corresponding portion of human NPP1 (e.g., over the length of the cysteine-rich region, the catalytic region, the c-terminal region, the cysteine-rich region plus the catalytic region, the cystein-rich region plus the catalytic region plus the c-terminal region. Preferred NPP1 variants have at least 90%, preferably at least 95%, more preferably at least 97% amino acid sequence identity to (i) the amino acid sequence of residues 205-591 of SEQ ID NO: 1, (ii) the amino acid sequence of residues 99-591 of SEQ ID NO:1, (iii) the amino acid sequence of residues 99-925 of SEQ ID NO:1, (iv) the amino acid sequence of residues 107-925 of SEQ ID NO:1, or (v) the amino acid sequence of residues 187-925 of SEQ ID NO:1. Suitable positions for amino acid variation are well-known from NPP1 structural studies and analysis of disease-associated mutations in NPP1. For example, substitution of the following amino acids occurs in certain disease-associated mutations that reduce NPP1 enzymatic activity, and variations of the amino acids at these positions should be avoided: Ser216, Gly242, Pro250, Gly266, Pro305, Arg349, Tyr371, Arg456, Tyr471, His500, Ser504, Tyr513, Asp538, Tyr570, Lys579, Gly586; Tyr659, Glu668, Cys726, Arg774, His777, Asn792, Asp804, Arg821, Arg888, and Tyr901. (See, e.g., Jansen, S. et al., Structure 20:1948-1959 (2012)).

In one embodiment, the soluble NPP1 protein can be a fusion protein recombinantly fused or chemically bonded (e.g., covalent bond, ionic bond, hydrophobic bond and Van der Waals force) to a fusion partner. In another embodiment, the fusion protein has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO:4. SEQ ID NO:4 is the amino acid sequence of sNPP1-Fc-D10 (SEQ ID NO: 4). The Fc sequence is underlined.

To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., sNPP1 amino acid sequence of SEQ ID NO:2; amino acids 107-925 of SEQ ID NO:1 or amino acids 187-925 of SEQ ID NO:1). The amino acid residues or nucleotides at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol Biol 1970, 48, 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 1989, 4, 11-17) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The sNPP1 can consist of or consist essentially of an NPP1 component as described herein. Alternatively, the sNPP1 can be in the form of a fusion protein that contains an NPP1 component and one or more other polypeptides, referred to as fusion partners, optionally through a suitable linker in each instance, or in the form of a conjugate between an NPP1 component and another molecule (e.g., PEG). When the sNPP1 is in the form of a fusion protein, each fusion partner is preferably located c-terminally to the NPP1 component. Without wishing to be bound by any particular theory, it is believed that fusion proteins that contain an NPP1 component that contains the cysteine-rich region and catalytic region, and one or more fusion proteins that are located c-terminally to the NPP1 component, are preferred over other configurations of NPP1 fusion proteins because they can be expressed at sufficient levels and are sufficiently stable to be used as therapeutic proteins.

Any suitable fusion partner can be included in the fusion protein. Advantageously, a number of fusion partners are well-known in the art that can provide certain advantages, such as reduced aggregation and immunogenicity, increased the solubility, improved expression and/or stability, and improved pharmacokinetic and/or pharmacodynamics performance. See, e.g., Strohl, W. R. BioDrugs 29:215-239 (2015). For example, it is well-known that albumin, albumin fragments or albumin variants (e.g., human serum albumin and fragments or variants thereof) can be incorporated into fusion proteins and that such fusion proteins can be easily purified, stable and have an improved plasma half-life. Suitable albumin, albumin fragments and albumin variants that can be used in the sNPP1 fusion proteins are disclosed, for example in WO 2005/077042A2 and WO 03/076567A2, each of which is incorporated herein by reference in its entirety. Fusions to human transferrin are also known to improve half-life. See, e.g., Kim B J et al., J Pharmacol Expr Ther 334(3):682-692 (2010); and WO 2000/020746. Peptides that bind to albumin or transferrin, such as antibodies or antibody fragments, can also be used. See, e.g., EP 0486525 B1, U.S. Pat. No. 6,267,964 B1, WO 04/001064A2, WO 02/076489A1, WO 01/45746, WO 2006/004603, and WO 2008/028977. Similarly immunoglobulin Fc fusion proteins are well-known. See, e.g., Czajkowsky D M et al., EMBO Mol Med 4(10):1015-1028 (2012), U.S. Pat. Nos. 7,902,151; and 7,858,297, the entire teachings of which are incorporated herein by reference in their entirety. The fusion protein can also include a CTP sequence (see also, Fares et al., Endocrinol 2010, 151, 4410-4417; Fares et al., Proc Natl Acad Sci 1992, 89, 4304-4308; and Furuhashi et al., Mol Endocrinol 1995, 9, 54-63). Preferably, the fusion partner is the Fc of an immunoglobulin (e.g., Fc or human IgG1). The Fc can include CH1, CH2 and CH3 of human IgG1, and optionally the human IgG1 hinge region (EPKSCDKTHTCPPCP (SEQ ID NO:13)) or a portion of the human IgG1 hinge region (e.g., DKTHTCPPCP (SEQ ID NO:14) or PKSCDKTHTCPPCP (SEQ ID NO:15)) if desired. In some fusion proteins, the Fc can include CH2 and CH3 of human IgG1, or the Fc of human IgG2 or human IgG4, if desired. Preferably, the sNPP1 fusion protein comprises an NPP1 component and a peptide that increases the half-life of the fusion protein, most preferably the Fc of an immunoglobulin (e.g., Fc or human IgG1). As used herein, a “protein that increases the half-life of the fusion protein” refers to a protein that, when fused to a soluble NPP1 or biologically active fragment, increases the half-life of the soluble NPP1 polypeptide or biologically active fragment as compared to the half-life of the soluble NPP1 polypeptide, alone, or the NPP1 biologically active fragment, alone. In one embodiment, the half-life of the NPP1 fusion protein is increased 50% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 60% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 70% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 80% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 90% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone.

In another embodiment, the half-life of the NPP1 fusion protein is increased 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. Methods for determining the half-life of a protein or fusion protein are well known in the art. For example, Zhou et al., Determining Protein Half-Lives, Methods in Molecular Biology 2004, 284, 67-77 discloses numerous methods for testing of the half-life of a protein. If desired, the fusion protein can be conjugated to polymers or other suitable compounds that extend half-life, such as polyethylene glycol (PEG), can be conjugated to the NPP1 fusion proteins.

In one embodiment, the peptide which increases the half-life of the fusion protein is a CTP sequence (see also, Fares et al., 2010, Endocrinol., 151(9):4410-4417; Fares et al., 1992, Proc. Natl. Acad. Sci, 89(10):4304-4308; and Furuhashi et al., 1995, Molec. Endocrinol., 9(1):54-63).

In another embodiment, the peptide which increases the half-life of the fusion protein is an Fc domain of an Ig.

Fusion partners may also be selected to target the fusion protein to desired sites of clinical or biological importance (e.g., site of calcification). For example, peptides that have high affinity to the bone are described in U.S. Pat. No. 7,323,542, the entire teachings of which are incorporated herein by reference. Peptides that can increase protein targeting to calcification sites can contain a consecutive stretch of at least about 4 acidic amino acids, for example, glutamic acids or aspartic acids. Typically, the peptide that targets the fusion protein to calcification sites will comprise between 4 and 20 consecutive acidic amino acids, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids selected from glutamic acid and aspartic acid. The peptide can consist solely of glutamic acid residues, solely of aspartic acid residues, or be a mixture of glutamic acid and aspartic acid residues. A particularly preferred moiety for targeting to sights of calcification is Asp₁₀ (SEQ ID NO:18).

In one embodiment, the NPP1 fusion protein of the invention comprises an NPP1 polypeptide and a moiety that increase protein targeting to calcification sites such as a consecutive stretch of acidic amino acids, for example, glutamic acids or aspartic acids.

Suitable peptide linkers for use in fusion proteins are well-known and typically adopt a flexible extended conformation and do not interfere with the function of the NPP1 component or the fusion partners. Peptide linker sequences may contain Gly, His, Asn and Ser residues in any combination. The useful peptide linkers include, without limitation, poly-Gly, poly-His, poly-Asn, or poly-Ser. Other near neutral amino acids, such as Thr and Ala can be also used in the linker sequence. Amino acid sequences which can be usefully employed as linkers include those disclosed in Maratea et al., Gene 1985, 40, 39-46; Murphy et al., Proc Natl Acad Sci USA 1986, 83, 8258-8262; U.S. Pat. Nos. 4,935,233 and 4,751,180. Other suitable linkers can be obtained from naturally occurring proteins, such as the hinge region of an immunoglobulin.

A preferred synthetic linker is (Gly₄Ser)_(n), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO:19). Preferably, n is 3 or 4. For example, in some embodiments the linker is (Gly₄Ser)₃ (SEQ ID NO:16) and the fusion protein include a linker with the amino acid sequence GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer (SEQ ID NO:16). Typically, the linker is from 1 to about 50 amino acid residues in length, or 1 to about 25 amino acids in length. Frequently, the linker is between about 8 and about 20 amino acids in length. Preferred NPP1 fusion proteins comprise from N-terminus to C-terminus an NPP1 component, optionally a linker, an Fc region of an immunoglobulin (e.g., human IgG1 Fc optionally including hinge or a portion thereof), optionally a second liner, and optionally a targeting moiety. Thus, the Fc region and the optional targeting moiety, when present, are each located C-terminally to the NPP1 component. The NPP1 component preferably comprises the cysteine-rich region and the catalytic domain of NPP1, lacks the N-terminal cytosolic and transmembrane domains, and optionally contains the C-terminal region.

A preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the C-terminal region of human NPP1; and the Fc region, including hinge, of a human immunoglobulin. Preferably, the Fc region is from human IgG1. In particular embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3. SEQ ID NO:3 is the amino acid sequence of sNPP1-Fc fusion protein.

A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO:3.

Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the C-terminal region of human NPP1; a linker (e.g., (Gly₄Ser)₃ (SEQ ID NO:16)); and the Fc region, including hinge, of a human immunoglobulin. Preferably, the Fc region is from human IgG1.

Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; the Fc region, including hinge or a portion thereof, of a human immunoglobulin; and a moiety targeting the fusion protein to sites of calcification. Preferably, the Fc region is from human IgG1. Preferably, the moiety targeting the fusion protein to sites of calcification is Asp₁₀ (SEQ ID NO:18). More preferably, the Fc region is from human IgG1 and the moiety targeting the fusion protein to sites of calcification is Asp₁₀ (SEQ ID NO:18). In particular embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4. A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO:4.

Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; a linker (e.g., (Gly₄Ser)₃ (SEQ ID NO:16)); the Fc region, including hinge or a portion thereof, of a human immunoglobulin; and a moiety targeting the fusion protein to sites of calcification. Preferably, the Fc region is from human IgG1. Preferably, the moiety targeting the fusion protein to sites of calcification is Asp₁₀ (SEQ ID NO:18). More preferably, the Fc region is from human IgG1 and the moiety targeting the fusion protein to sites of calcification is Asp₁₀ (SEQ ID NO:18).

Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising a portion of the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; optionally a linker (e.g., (Gly₄Ser)₃ (SEQ ID NO:16)); the Fc region, including hinge or a portion thereof, of a human immunoglobulin. Preferably, the Fc region is from human IgG1. In particular embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Preferred fusion protein of this type have the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. In particularly preferred aspects, a fusion protein of SEQ ID NO:3 is administered in accordance with the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:4 is administered in accordance with in the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:9 is administered in accordance with in the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:10 is administered in accordance with the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:11 is administered in accordance with the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:12 is administered in accordance with the methods described herein.

Fusion proteins of the present invention can be prepared using standard methods, including recombinant techniques or chemical conjugation well known in the art. Techniques useful for isolating and characterizing the nucleic acids and proteins of the present invention are well known to those of skill in the art and standard molecular biology and biochemical manuals can be consulted to select suitable protocols for use without undue experimentation. See, for example, Sambrook et al., 1989, “Molecular Cloning: A Laboratory Manual”, 2^(nd) ed., Cold Spring Harbor, the content of which is herein incorporated by reference in its entirety.

The isolated recombinant human sNPP1, fragment, and fusion proteins thereof, can be produced in any useful protein expression system including, without limitation, cell culture (e.g., CHO cells, COS cells, HEK203), bacteria such as Escherichia coli (E. coli) and transgenic animals, including, but no limited to, mammals and avians (e.g., chickens, quail, duck and turkey). For expression, a construct that encodes the sNPP1 and includes a suitable signal sequence (e.g., from human Ig heavy chain, NPP2, NPP4, NPP7 or human serum albumin, for example) in frame with the sequence of the sNPP1 and operably linked to suitable expression control elements.

The sNPP1, including the fusion proteins, and physiologically acceptable salt forms thereof are typically formulated into a pharmaceutical composition for administration in accordance with the methods described herein. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier or excipient. Compositions comprising such carriers, including composite molecules, are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 14^(th) ed., Mack Publishing Co., Easton, Pa.), the entire teachings of which are incorporated herein by reference. The carrier may comprise a diluent. In one embodiment, the pharmaceutical carrier can be a liquid and the fusion protein may be in the form of a solution. The pharmaceutical carrier can be wax, fat, or alcohol. In another embodiment, the pharmaceutically acceptable carrier may be a solid in the form of a powder, a lyophilized powder, or a tablet. In one embodiment, the carrier may comprise a liposome or a microcapsule. The pharmaceutical compositions can be in the form of a sterile lyophilized powder for injection upon reconstitution with a diluent. The diluent can be water for injection, bacteriostatic water for injection, or sterile saline. The lyophilized powder may be produced by freeze drying a solution of the fusion protein to produce the protein in dry form. As is known in the art, the lyophilized protein generally has increased stability and a longer shelf life than a liquid solution of the protein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the any plurality of the dependent claims or Examples is contemplated to be within the scope of the disclosure.

INCORPORATION BY REFERENCE

The disclosure of each and every U.S. and foreign patent and pending patent application and publication referred to herein is specifically incorporated herein by reference in its entirety, as are the contents of Sequence Listing and Figures.

EXAMPLES

The present invention is further exemplified by the following examples. The examples are for illustrative purpose only and are not intended, nor should they be construed as limiting the invention in any manner.

Example 1: Human Primary Vascular Smooth Muscle Cells (VSMCs)

To assess whether ENPP1 knockdown increases proliferation in human primary vascular smooth muscle cells (VSMCs) the following experiments were conducted using human primary VSMCs obtained from ATCC and ThermoFisher Scientific.

1A. ENPP1 Gene Expression

First, baseline ENPP1 gene expression in human VSMCs was assessed via real time polymerase chain reaction (qRT-PCR), western blot analysis, and an assay to detect cell based ENPP1 enzymatic activity, according to the following protocols.

Real Time Polymerase Chain Reaction (qRT-PCR): Total RNA was isolated from human VSMCs using a Qiagen Rneasy Mini kit (cat #74106) and QIAshredder (cat #79656, QIAGEN, Valencia, Calif.) as per manufacturer's instructions. The isolated RNA was quantified using a Nanodrop2000 (Thermoscientific) and reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Cat #4368814; ThermoFisher Scientific). The resulting cDNA was amplified using the TaqMan Universal PCR Master Mix and detected by real-time PCR using QuantStudio™ 7 Flex System. TaqMan probes for human ENPP1, Hs01054038_m1 and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs99999905_m1 were obtained from ThermoFisher Scientific. Target gene expression level was normalized by GAPDH level in each sample and Relative expression level was calculated using 2-ΔΔ Ct method.

1B. Western Blot Analysis

VSMCs were detached, washed in PBS and the cell lysates were prepared in lysis buffer containing 1% each of protease inhibitor, Phosphatase Inhibitor Cocktail 3 (cat #P0044; Sigma) and Phosphatase Inhibitor Cocktail 2 (cat #P5726; Sigma). The cell lysate was quantified and denatured, then equal amounts of the protein were loaded on 4-12% Bis polyacrylamide gels. The proteins in gels were electrophoretically transferred to nitrocellulose membrane using iBlot® 2 Dry Blotting System. Following treatment with blocking buffer (5% skimmed milk in 1XTBST (Cat #IBB-180; Boston Bioproducts)), and incubated with Rabbit pAb to human ENPP1 (PA527905 by Thermo Fischer Scientific) at 1:500 or GAPDH (14C10) Rabbit anti GAPDH mAB by Cell Signaling Technology (cat #2118L) at 1:1000 in blocking buffer for overnight at 4° C., followed by a Goat-anti-Rabbit Antibody conjugated with HRP (cat #7074; Cell signaling technology) for 1 hr. at room temperature. The signals were detected using the Protein Simple FluorChem R system (Part #92-15313-00, ProteinSimple). Signals of ENPP1 protein were normalized with level of endogenous protein GAPDH in each sample.

1C. Cell Based ENPP1 Enzymatic Activity

Cell based ENPP1 enzyme activity was assayed using the colorimetric substrate, p-nitrophenyl thymidine 5′-monophosphate (Cat #T4510, Sigma). Cells were seeded into the well of 96 well maxisorp plate at 300,000 cells/well and 100,000 cells/well and substrate at a final concentration of 1 mM in reaction buffer (200 mM Tris-HCl pH9, 1M NaCl, 10 mM MgCl2, 0.1% (v/v) Triton-X 100) was added to the plate. Enzyme activity is measured the reaction product based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol with absorbance at 405 nm using a continuous spectrophotometric assay using in a FlexStation® Plate Reader (Molecular Devices) in a kinetic mode with 21 reads at 31 sec intervals. Standard curve was generated using ENPP1-Fc protein ranged from 0 ng/ml to 90 ng/ml. Data was analyzed at 10 minutes respectively.

Results: FIG. 1 depicts mRNA (FIG. 1A) and protein (FIG. 1B) ENPP1 expression in human VSMCs from six different donors, as well as enzyme activity from three of the donors (FIG. 1C). As shown in FIG. 1, there was significant natural variability in baseline ENPP1 expression in human primary VSMCs.

1D. ENPP1 Knockdown Using siRNA Targeting Human ENPP1

Human VSMCs (donor 3) were transfected with either ENPP1 siRNA or control siRNA. The following siRNA constructs were used: 1 (ENPPA (CDS) Location: 825), 2 (ENPPA (CDS) Location: 813), 3 (ENPPA (CDS) Location: 1272), 4 (ENPPA (CDS) Location: 447), and 5 (ENPPA (CDS) Location: 444). Human VSMCs (donor 3, passage 4) were transfected with either siRNA targets [Silencer Select ENPP1 siRNA #1: s10264 (Cat #4390824), siRNA #2 s10265 (Cat #4390824), siRNA #3: s10266 (Cat #4390824), siRNA #4: s224228 (Cat #4392420), Silencer ENPP1 siRNA #5: 144240 (Cat #AM90824); Life Technologies] to human ENPP1 or control using the Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) following the manufacturer's instructions. Cells were seeded in 60 mm dish at density 3500 cells/0.32 cm in complete medium (Vascular Cell Basal medium ATCC #PCS-100-030 supplemented with Vascular Smooth Muscle Cell Growth Kit ATCC #PCS-100-042, ATCC). Cells were treated with either an siRNA that specifically targets human ENPP1 or a negative control siRNA at a concentration of 100 nM in OPTI-MEM (cat #31985; ThermoFisher Scientific) and incubated at 37° C. Cells were harvested 48 hours or 6 days or 11 days after transfection (cells for 6 day and 11 day time points were harvested and reseeded at 48 hours), total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR. Levels of ENPP1 mRNA expression are reported as percentage of inhibition in mRNA expression relative to negative-siRNA after normalization to GAPDH mRNA levels.

Results: As shown in FIG. 2A, ENPP1 mRNA expression was inhibited by 90% or greater relative to the negative control for all five siRNA constructs, 48 hours post siRNA transfection. Specifically, ENPP1 mRNA expression was inhibited by 91.3% using siRNA construct #1, 93.1% using siRNA construct #2, 94% using siRNA construct #3, 93.6% using siRNA construct #4, and 90.2% using siRNA construct #5, relative to the negative control. Moreover, as shown in FIG. 2B, ENPP1 mRNA expression was inhibited by 84.2% six days post siRNA transfection with construct #4 (relative to the negative control) and 74.6% eleven days post siRNA transfection with construct #4 (relative to the negative control). Accordingly, the data indicates that siRNA sufficiently silences ENPP1 expression for a prolonged period of time.

1E. Effect of ENPP1 Knockdown on Proliferation

Human VSMCs (Donor 1 and Donor 3, passage 4) were seeded in 60 mm dishes at density 0.3*10e6 cells/60 mm dish in Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). After overnight recovery, they were transfected with ENPP1 siRNA or control siRNA in OPTI-MEM. After 48 hours, cells were harvested and reseeded at 2500 cells/well into 96 well plate. Cells were cultured in basal medium containing 2% or 5% FBS. Cell proliferation was evaluated by [3H] thymidine uptake. [3H]-thymidine was added in the last 18 hours of culture. Results are expressed as CPM±SEM. Experiments were triplicated.

Results: As shown in FIGS. 3A (Donor 1) and 3B (Donor 3) and Table 1 (Donor 3), silencing of ENPP1 by siRNA increased proliferation of human primary VSMCs from these two different donors (e.g., by about 1.75 fold or greater) compared to negative control siRNA.

TABLE 1 Inhibition of ENPP1 siRNA ENPP1 (CDS) at 48 hrs. siRNA ENPP1 813 84.7% siRNA_NC N/A 0.0%

1F. Effect of ENPP1 Knockdown on Cell Proliferation

Human VSMCs (Donor 1 and Donor 3, passage 4) were seeded in 60 mm dishes at density 0.3*10e6 cells/60 mm dish in Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). After overnight recovery, they were transfected with ENPP1 siRNA or control siRNA in OPTI-MEM. After 48 hours, cells were harvested and reseeded at 2500 cells/well into 96 well plate. Cells were cultured in basal medium containing 5% FBS for 3 or 4 days. Cells were detached and stained with AOPI at indicated time point, cell number was measured using auto cell counter Cellometer Auto 2000. Results were expressed as Cell number ±SEM. Experiments were triplicated.

Results: As shown in FIGS. 4A (Day 3) and 4B (Day 4) representing Donor 3, silencing ENPP1 using siRNA increased cell growth in human primary VSMCs. Specifically, cell growth was at least two fold or greater 3-4 days after silencing ENPP1 using two different constructs. These results are consistent in light of results found from independent experiments, independent analysts, different constructs, same donor, and different methods.

Example 2: Rat Primary Vascular Smooth Muscle Cells (VSMCs)

To assess whether ENPP1 knockdown increases proliferation, the following experiments were conducted using rat primary VSMCs.

First, an in vitro primary rat VSMC Model system was established. Primary rat vascular smooth muscle cells (VSMCs) were prepared by using enzymatic digestion of thoracic arteries from 3-week-old Sprague-Dawley rats. Small fragments were minced and digested at 37° C. in vascular cell basal media (ATCC) supplemented collagenase type II 2 mg/ml (Cat #17101-015, Gibco) for 3 hours, mix every 15 minutes and replace digestion solution hourly. Then, the cell suspension was centrifuged at 1000 rpm for 10 min at 4° C., the pellet was resuspended in complete medium and cultured into T75 flasks. Cells were cultured in vascular cell basal media (ATCC #PCS-100-030) containing 5% fetal bovine serum, and growth supplements (Cat #PCS-100-042, ATCC containing with 5 ng/ml rhFGF, 5 μg/ml rh Insulin, 50 μg/ml Ascorbic acid, 10 mM L-gutamine, 5 ng/ml rhEGF, penicillin 10 Units/ml, streptomycin 10 μg/ml, and Amphotercin B 25 ng/ml). VSMCs were subcultured and used between passages 3-4. ENPP1 knockdown was achieved by transfection with siRNA. Specifically, transfection of VSMCs with siRNA was performed using Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) according to the manufacturer's instructions.

2A. Effect of Silencing ENPP1 on Pharmacological Activity

Rat primary VSMCs (passage 3) were seeded into 60 mm dish at density 0.3*10e6/60 mm dish in complete medium and transfected with either one of a siRNA specific targets to rat ENPP1 or negative control siRNA at a concentration of 100 nM in OPTI-MEM (cat #31985; ThermoFisher Scientific) and incubated at 37° C. Cells were harvested at 48 hours and reseeded in the wells of 6 well plate at 37500 cells/well in complete medium. Cells were then harvested at indicated time points after transfection, total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR. Levels of ENPP1 mRNA expression are reported as percentage of inhibition in mRNA expression relative to negative-siRNA after normalization to GAPDH mRNA levels (see FIG. 5A).

Rat VSMCs (passage 3) were transfected with either ENPP1 siRNA or control siRNA for 48 hours, then seeded into the wells of 6-well plate at 37500 cells/well (2 wells per condition), the cells were stimulated with Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). Cells were detached at indicated time points and stained with AOPI, cell number was measured using auto cell counter, Cellometer Auto 2000. Results are expressed as Cell number ±SEM (see FIG. 5B). Experiments were triplicated.

ENPP1 enzyme activity was assayed using the colorimetric substrate, p-nitrophenyl thymidine 5′-monophosphate (Cat #T4510, Sigma). Cells were seeded into the well of 96 well plate at 20000 cells/well and substrate at 1 mM p-nitrophenyl thymidine 5′-monophosphate in reaction buffer was added. Enzyme activity was measured the reaction product based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol with absorbance at 405 nm using a continuous spectrophotometric assay using in a FlexStation® Plate Reader (Molecular Devices) in a kinetic mode with 21 reads at 31 sec intervals. Standard curve was generated using ENPP1-Fc protein ranged from Ong/ml to 90 ng/ml. Data was analyzed using SoftMax Pro software at 10 minutes. ENPP1 activity in each sample was calculated based on the standards (see FIG. 5C).

As shown in FIGS. 5A-5C, siRNA silencing of ENPP1 was robust and durable (FIG. 5A), increased cell growth (FIG. 5B), and decreased enzyme activity (FIG. 5C).

2B. Effect of Adenosine, AMP, or PPi on Proliferation of Rat VSMCs

Rat VSMCs (p3) were transfected with either rat ENPP1-siRNA or control siRNA for 48 hrs and then seeded into wells of 96-well plate at 2500 cells/well. Cells were then cultured in complete medium in the presence and absence of Adenosine monophosphate (AMP) (Cat #A1752, Sigma), adenosine (A4036, Sigma) or PPi (Cat #71515, Sigma). Cell proliferation was evaluated on day 3 by [3H] thymidine uptake. Results are expressed as CPM±SEM. Experiments were triplicated.

As shown in FIG. 6A, adenosine and AMP inhibited proliferation in rat VSMCs that were knocked down with ENPP1 and without regulated ENPP1. However, PPi did not affect proliferation in rat VSMCs (FIG. 6B).

2C. Effect of Bisphosphonate on Proliferation of Rat VSMCs

Rat VSMCs (p3) were transfected with either rat ENPP1-siRNA or control siRNA for 48 hrs., then seeded into wells of 96-well plate at 2500 cells/well, cells were cultured in complete medium in the presence and absence of Etidronate (Cat #P5248, Sigma) at indicated concentration. Cell proliferation was evaluated on day 3 by [3H] thymidine uptake. Results are expressed as CPM±SEM. Experiments were triplicated. Data was reproducible with Zoledronate as well.

As shown in FIG. 7, bisphosphonate did not appear to inhibit proliferation in rat VSMCs.

2D. Effect of Silencing ENPP1 on Proliferation of Rat VSMCs

Rat VSMCs were transfected with siRNA against rat ENPP1 si206 (ENPP1 sequence start position: 462; (Cat #SASI_Rn01_00111206 NM_053535, Sigma)), rat ENPP1 sil53 (ENPP1 sequence start position: 516 (Cat #SASI_RnO2_00266153 NM_053535, Sigma)), or a negative control (FIG. 8A). After 48 hours transfection, cells were seeded into wells of 96-well plate at 1250 cells/well in complete medium. After 4 hours, following treatment conditions were added in complete medium: PDGF (Cat #P8953, Sigma), Cilostazol (Cat #0737, Sigma), PDGF+Cilostazol at indicated concentration. [3H] thymidine was added in the last 18 hours of culture. Cell proliferation was evaluated by [3H] thymidine uptake. [3H]-thymidine was added in the last 18 hours of culture. Results are expressed as CPM±SEM. Experiments were triplicated.

As shown in FIGS. 8A-8B, silencing ENPP1 by siRNA increased proliferation in rat VSMCs, but was inconsistent between constructs.

Example 3: Overexpression of Mouse or Rat ENPP1 in Rat Primary Vascular Smooth Muscle Cells (VSMCs)

Experiments were conducted to assess whether overexpressed ENPP1 rescues proliferation of VSMCs. The following constructs were used: (1) siRNA target rENPP1:(SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma, (2) Ad-mENPP1: Vector Biolab (Lot #20150616T #10; Vector Biolabs), (3) Ad-rENPP1: Life Tech+Vector Biolab (Lot #20150714T #11; Vector Biolabs), and (4) Ad-rENPP1: GeneWiz+Vector Biolab (Lot #20150714T #9; Vector Biolabs).

3A. Ad-mENPP1/Ad-rENPP1 Induces Overexpression of Mouse/Rat ENPP1 mRNA Specifically in Rat VSMC

Co-transfection with Ad-GFP and siRNA: Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector Ad-GFP (cat #1060, Vector Biolabs) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs. at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. The efficacy of adenovirus infectivity was measured 45 hours after infection of Ad-GFP under a fluorescent microscope (DMI8 Leica Microsystems) at 100× magnification.

Co-transfection with Ad-mENPP1 and siRNA-NC: The primary Rat Vascular Smooth Muscle Cells (Rat VSMC) (Passage 3) were seeded at 6000 cells/0.32 cm2 in a 6 well dish in complete medium (Vascular Cell Basal medium ATCC #PCS-100-030 supplemented with Vascular Smooth Muscle Cell Growth Kit ATCC #PCS-100-042). After overnight recovery of the cells, the cells were infected with siRNA particles using Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) diluted in OPTI-MEM (cat #31985; ThermoFisher Scientific) containing 0.25% FBS. After 4 hours, remove the media and add the adenoviral particles at MOI=400. Three adenoviral particles tested are Ad-rENPP1 (Lot #20150714T #9; Vector Biolabs), Ad-rENPP1 (Lot #20150714T #11; Vector Biolabs) and Ad-mENPP1 (Lot #20150616T #10; Vector Biolabs). The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Cells were harvested 48 hours after infection with Ad-mENPP1. Total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR using primer specific to mouse ENPP1 or rat ENPP1. Levels of mENPP1 mRNA expression are reported as percentage of mRNA expression relative to negative control Ad after normalization to GAPDH mRNA levels.

Real Time Polymerase Chain Reaction (qRT-PCR): RNA isolation and reverse transcription were performed using the TaqMan® Gene Expression Cells-to-CT™ Kit (Cat #AM1729, Thermofisher Scientific) as per manufacturer's instructions in a 96-well plate format, 96 hours post co-transfection. The resulting cDNA is amplified using the TaqMan Universal PCR Master Mix and detected by real-time PCR using QuantStudio™ 7 Flex System in a 384 well-plate. TaqMan probes for rat ENPP1 (AJKAK71), Menpp1 (Mm00501088_m1) and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Rn01775763_g1 were obtained from ThermoFisher Scientific. Target gene expression level was normalized by GAPDH level in each sample and Relative expression level was calculated using Δ Ct method.

The Rat ENPP1 (AJKAK71) taqman probe was custom designed to detect the rENPP1 in the adenoviral vector cassette while the mouse ENPP1 (Mm00501088_m1) taqman probe was a premade probe from ThermoFisher Scientific.

As shown in FIG. 9A, Ad-mENPP1/Ad-rENPP1 induced overexpression of Mouse/Rat ENPP1 messenger RNA specifically in rat VSMC. Nearly 100% of VSMCs expressed high level GFP 45 hrs. after co-transfection with siRNA and Ad-GFP (FIG. 9B). Moreover, there was specific overexpression of mouse and rat ENPP1 in co-transfected Rat VSMC starting at the 48 hour timepoint, which and persisted through 96 hours.

3B. mENPP1 mRNA Over-Expression Persists in Presence of siRNA Targeting Rat ENPP1, but with Moderate Interference of Mouse ENPP1

Co-transfection with Ad-ENPP1 and siRNA: Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm² in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-mENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Cells were harvested 48 hours after infection of Ad-mENPP1. Total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR. Levels of ENPP1 mRNA expression are reported as percentage of mRNA expression relative to negative-siRNA after normalization to GAPDH mRNA levels. Ps: siNC (Silencer Select ENPP1 siRNA s10265 (5 nmol)) Cat #4390824, ThermoFisher Scientific. siRNARat enpp1 (SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma.

As shown in FIG. 10, mENPP1 mRNA overexpression persisted in the presence of siRNA targeting rat ENPP1, but with moderate interference of mouse ENPP1. Mouse ENPP1 exhibited 92% homology to rat ENPP1 at the gene level. Accordingly, siRNA knock down of rat ENPP1 expression also partially down regulated mouse ENPP1 expression.

3C. Successful Rescue ENPP1 Protein Expression by Ad-rENPP1

Co-transfection with Ad-ENPP1 and siRNA: Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-rENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Cells were harvested 72 hours after infection of Ad-rENPP1. ENPP1 protein levels were measured using in cell western blot assay.

In cell western: The cells were fixed in 4% Formaldehyde (w/v), Methanol-free (cat #28908, Pierce™) for 20 min at room temperature. The formaldehyde was removed under a fume hood and the cells were washed twice with 200 μl of PBS. The cells were permeabilized in 100p/well (Cat #3603, Costar) of 0.1% Triton x-100 in PBS for 20 min at Room temperature. Then, the cells were blocked using LICOR TBS blocking buffer (P/N 927-50000) for 1 hr. at Room Temperature followed by overnight incubation at 4° C. with 2.5 μg/ml primary antibody Goat anti-ENPP1/PC1 (Cat #OAEB02445, Aviva) in LICOR TBS blocking buffer containing 0.2% Tween20. The wells were washed thrice with 200 μl of 1×TBST and incubated with secondary antibody IRDye® 800CW Donkey-anti-Goat (P/N 926-32214) Antibody at 1:1000 for 1 hr. at Room Temperature covered in foil. The wells were washed thrice with 200 μl of 1×TBST and rinsed with TBS once to get rid of tween. Image the plate in LICOR Odessey Clx.

As shown in FIG. 11, ENPP1 protein expression was successfully rescued by Ad-rENPP1.

3D. Overexpression of mENPP1 Rescues Enzyme Activity in rVSMCs and A10 Cells

Co-transfection with Ad-ENPP1 and siRNA: Rat VSMCs (passage 3) (upper) or rat non-differentiated VSMCs A-10 cells (ATCC, CRL-1476) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-rENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Cells were harvested 72 hours after infection of Ad-rENPP1. Cell based ENPP1 enzyme activity was using the colorimetric substrate, p-nitrophenyl thymidine 5′-monophosphate (Cat #T4510, Sigma). Cells were seeded into the well of 96 well Maxisorp plate at 20000 cells/well and substrate at 1 mM p-nitrophenyl thymidine 5′-monophosphate in reaction buffer was added. Enzyme activity is measured the reaction product based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol with absorbance at 405 nm using a continuous spectrophotometric assay using in a FlexStation® Plate Reader (Molecular Devices) in a kinetic mode with 21 reads at 31 sec intervals. Standard curve was generated using ENPP1-Fc protein ranged from Ong/ml to 90 ng/ml. Data was analyzed using SoftMax Pro software at 10 minutes. ENPP1 activities in each sample was calculated based on the standards. siNC (Silencer Select ENPP1 siRNA s10265 (5 nmol)) Cat #4390824, ThermoFisher Scientific siRNA Rat enpp1 (SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma.

As shown in FIGS. 12A-12B, over-expression of mENPP1 rescued enzyme activity in rVSMCs and A10 cells (nondifferentiated rat VSMCs). Rat ENPP1 activity was not detected in A10 cells, but, low enzyme activity was observed in primary VSMCS transfected with Ad-rENPP1 (GeneWiz).

3E. Silencing ENPP1 Increases Proliferation in Rat VSMCs, Whereas Over-Expression of Mouse or Rat ENPP1 Inhibits Proliferation

Co-transfection with Ad-ENPP1 and siRNA: Rat VSMCs (passage 3) were seeded in the wells of 96-well plate at 6000 cells/well in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector Ad-rENPP1 or Ad-mENPP1 was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hours at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Day 1 post transfection, starvation media (0.25% FBS in basal media) was added to the starvation condition, complete media was added to non-starvation condition.

Forty eight hours post transfection, complete media was added to starvation condition cells. Cell proliferation was evaluated at Day 4 post-transfection by [3H] thymidine uptake. [3H] thymidine was added in the last 18 hours of culture (see FIGS. 13A-13B).

In a separate experiment, forty eight hours post transfection, cells were seeded into wells of 24-well plate at 15000 cells/well in complete medium for 4 hours, followed by 48 hours starvation in 0.25% FBS, the cells were cultured in base media contains 5% FBS (lower). Cells were stained with AOPI and counted using auto cell counter 72 hours later (See FIG. 14 and Table 2) siNC (Silencer Select ENPP1 siRNA s10265 (5 nmol)) Cat #4390824, ThermoFisher Scientific siRNA Rat enpp1 (SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma.

Results are expressed as Cell number SEM. Experiments were triplicated.

TABLE 2 Sample ID Viability (%) siNC + AdNC 89.35 si-rENPP1 + AdNC 85.2 si-rENPP1 + AdmENPP1 89.25 si-rENPP1 + AdrENPP1 85.9

As shown in FIGS. 13A-13B and FIG. 14, silencing ENPP1 increased proliferation in rat VSMCs, whereas overexpression of mouse or rat ENPP1 inhibited proliferation.

In summary, the above experiments demonstrated that silencing ENPP1 increased proliferation and cell growth of VSMCs in all tested systems. Moreover, over expression of mouse or rat ENPP1 using an Ad vector inhibited proliferation and cell growth in rat VSMCs.

Example 4: In Vitro Proof of Concept in Rat Primary VSMCs

Rat VSMCs were seeded in 35 mm dish at density 75000 cells/9.6 cm² in completed medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 or siRNA negative control (siNC) for overnight. The cells were then starved with base medium contains 0.25% FBS. After 24 hrs starvation, the cells were reseeded into the well of 96 well plates at 2500 cells/0.32 cm2 in CM. After 4 hrs, once the cells adhered, ATP treatments (FLAAS, Sigma) were added at 1M in final concentration. After 30 minutes, 100 μl of supernatant was collected and tested via CellTiter-Glo® Luminescent Cell Viability Assay (cat #G7572, Promega) in a black/opaque plate. Also, cell titer glo with the seeded cells was performed per manufacturer's instruction. This protocol was repeated 2 hours and 24 hours post ATP treatment.

As shown in FIG. 15 (30 minute), FIG. 16 (2 hour), and FIG. 17 (24 hour), ATP is unstable in complete medium containing 5% FBS in culture at 37° C. Moreover, as shown in FIG. 18 heat denatured human/mouse ENPP1-Fc protein completely lost enzymatic activity.

In a separate experiment, rat VSMCs were seeded in the well of the 6-well plates in complete medium contains 5% FBS. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (SASI_Rn01_00111206 Cat #PDSIRNA2D, Sigma) or siRNA negative control (Silencer Select ENPP1 siRNA s10265 (5 nmol) Cat #4390824, ThermoFisher Scientific) for overnight. The cells were then starved with base medium contains 0.25% FBS. After 24 hrs starvation, the cells were reseeded into the well of 96 well plates and cultured with completed medium contains 5% FBS in presence with 300 μM ATP and mENPP1-Fc protein (FIG. 19A), hENPP1-Fc (FIG. 19B), or hENPP1-Fc-D10 (FIG. 19C) protein, at the indicated concentration. The cultured medium was replaced daily. Proliferation was measured at day 3 using MicroBeta 3H-Thymidine incorporation. Thymidine was added in the last 18 hours.

As shown in FIGS. 19A-C, treatment with any of the ENPP1 proteins (mENPP1-Fc (FIG. 19A), hENPP1-Fc (FIG. 19B), and hENPP1-Fc-D10 (FIG. 19C)) inhibited proliferation on rat primary VSMCs. Heat denatured ENPP1 had no effect on proliferation of rat VSMCs.

Example 5: Human Induced Pluripotent Stem Cell (hiPSC)-Derived Vascular Smooth Muscle Cells (iVSMCs)

The following experiments were conducted using human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs).

5A. ENPP1 Expression

Human iPSC derived VSMCs (donors BJ, BLS) and human primary VSMCs (donors 1, 3, and 6) were cultured in T75 flasks in iVSMC cultured medium and complete medium, respectively. Cells were harvested at 80% confluence, total RNA was isolated from cells using a Qiagen Rneasy Mini kit (cat #74106) and QIAshredder (cat #79656, QIAGEN, Valencia, Calif.) as per manufacturer's instructions. The isolated RNA was quantified using a Nanodrop2000 (Thermoscientific) and reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Cat #4368814; ThermoFisher Scientific). The resulting cDNA is amplified using the TaqMan Universal PCR Master Mix and detected by real-time PCR using QuantStudio™ 7 Flex System. TaqMan probes for human ENPP1, Hs01054038_m1 and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs99999905_m1 were obtained from ThermoFisher Scientific. Target gene expression level was normalized by GAPDH level in each sample and Relative expression level was calculated using 2-ΔΔ Ct method (FIG. 20A).

Western Blot Analysis: The VSMCs were detached, washed in PBS and the cell lysates were prepared in lysis buffer containing 1% each of protease inhibitor (p8340; Sigma), Phosphatase Inhibitor Cocktail 3 (cat #P0044; Sigma) and Phosphatase Inhibitor Cocktail 2 (cat #P5726; Sigma). The cell lysate was quantified and denatured, then equal amounts of the protein were loaded on 4-12% Bis polyacrylamide gels. The proteins level was measured once the gel was electrophoretically transferred to nitrocellulose membrane using iBlot® 2 Dry Blotting System. The membranes were blocked for 1 h in blocking buffer (Licor TBS blocking buffer (P/N 927-50000) and incubated with Rabbit pAb to human ENPP1 (PA527905 by Thermo Fischer Scientific) at 1:500 and GAPDH (14C10) Rabbit anti GAPDH mAB by Cell Signalling Technology (cat #2118L) at 1:1000 in blocking buffer containing 0.2% Tween20 overnight at 4° C., followed by a Donkey-anti-Rabbit Antibody conjugated with the fluorescent dye IRDye® 800CW (cat #926-32213; LICOR) or Donkey-anti-Rabbit Antibody conjugated with the fluorescent dye IRDye® 680RD (cat #926-68073; LICOR) for 1 h at room temperature, respectively. The signals were detected using the Odyssey CLx Imaging System (LI-COR Biosciences). Signals of ENPP1 protein were normalized with level of endogenous protein GAPDH in each sample. The relative protein expression for the human primary iVSMC donors and the VCMC donors are depicted in FIGS. 20B and 20C, respectively.

As shown in FIGS. 20A-B, human primary iVSMCs expressed a high level of ENPP1.

5B. Effect of Silencing ENPP1 on Growth of Human iVSMCs.

Human iPSC differentiated VSMCs (iVSMCs) (p2) were seeded into the wells of 6-well plate and transfected with ENPP1 siRNA or negative control siRNAs, either consisting of a scrambled nucleotide sequence or directed to actin. After 48 hours transfection, the cells were detached, stained with AOPI and counted in auto cell counter Cellometer 2000.

As shown in FIG. 21, silencing ENPP1 by siRNA increased the growth of human iVSMCs as compared to both of the negative controls.

5C. In Vitro Proof of Concept Human iVSMCs

Additional experiments were conducted using human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs) to assess the effect of murine and human ENPP1 protein on proliferation. The following ENPP1 proteins were used: mENPP1-mG1FC: ZLC022, hENPP1-FC: 105-FC, and hENPP1-FC-D10: 105-FC-D10.

The human iPSC derived VSMCs were seeded at 3500 cells/0.32 cm2 in collagen 1 coated 60 mm dishes in SmGM-2 Smooth Muscle Growth Medium-2 (Cat #CC-3182, Lonza). After overnight culture, the cells were transfected with siRNA targets to human ENPP1 using Lipofectamine RNAiMAX (cat #13778500, ThermoFisher Scientific) for overnight. Following 48 hours starvation with 0.25% FBS, cells were stained with AOPI and counted with auto cell counter, Cellometer 2000. The effect of ENPP1-Fc protein on proliferation in VSMCs was measured using 3H thymine incorporation. Cells were reseeded in the wells of a 96 well plate that was pre-coated with collagen 1 in completed medium presence with 300 μM ATP in addition to absence or presence of varied concentration of mENPP1-Fc, hENPP1-Fc, or hENPP1-FC-D10 protein for 3 days. Culture medium was replaced daily. 3H thymine was added in the last 18 hours of culture. Results presents as mean±SEM of four wells (n=4). A similar pattern was confirmed in three independent assays. The statistical analysis was performed by student T test.

As shown in FIGS. 22A-C, all of the ENPP1 proteins significantly inhibited proliferation in human iVSMCs at all concentrations tested.

5D. PPi Assay Using Human iVSMCs

PPi is produced in an ENPP1 enzyme catalyzed reaction. PPi level was measured in culture supernatant using a radioactive assay. In order to avoid precipitation of magnesium pyrophosphate, assay components were divided into stock solutions. First, a master mix was prepared with 49.6 mM Trizma Acetate (cat #93337; Sigma-Fluka), 4.5 mM Magnesium acetate tetrahydrate (cat #63049; Sigma-Fluka); 3.5 μM NAPD-Na2 (cat #10128058001; Roche); 16.2 μM D-Glucose-1,6-diphosphate (cat #G6893; Sigma); 6.6 μM Uridine-5-Diphosphoglucose (cat #U4625; Sigma); 0.002 U/l Phosphoglucomutase (cat #P3397; Sigma); 0.003 U/l Glucose-6-phosphate dehydrogenase (cat #10165875001; Roche) and MilliQ water. Add 0.00118 U/l Uridine 5′-diphosphoglucose pyrophosphorylase (cat #U8501; Sigma) and 0.0002 μCi/μl Uridine diphospho-D-[6-3H] glucose (cat #NET1163; Perkin Elmer) to the master mix right before adding to the samples, and 25 μl of sample was added into 115 μl master mix. After 30 minutes incubation at 37° C., 200 μl of cold 3% activated charcoal (cat #C5510; Sigma) was added and incubated 30 min at 4° C. with vortexing every 10 min intervals. The contents of the tube was transferred into a 96 well filter plate (cat #8130; Pall Corporation). The reaction mix was filtered and 80 μl of supernatant was transferred to Iso beta plates (cat #6005040; Perkin Elmer) keeping the same plate position using 960 LTS Wide-orifice tips in 10 racks, presterilized. Add 120 μl of scintillation liquid Ultima Gold (cat #6013321; Perkin Elmer) and mixed well. The plated were incubated for 1 hour and then read via a micro beta counter.

As shown in FIG. 23, PPi was detected in the supernatant collected from human iVSMCs treated with mENPP1-Fc. Similar results were obtained using both hENPP1-Fc and hENPP1-Fc-D10.

In summary, all three ENPP1 proteins (mENPP1-Fc, hENPP1-Fc, and hENPP1-Fc-D10) significantly inhibited proliferation in human iVSMCs that silenced ENPP1. Heat denatured ENPP1 had no effect on proliferation of iVSMCs. The inhibition was more potent in human iVSMC than it was in rat VSMCs.

5E. Effect of Bisphosphonate on Proliferation of Human iVSMCs

The effect of Bisphosphonate on proliferation in human iVSMCs that silenced ENPP1 was evaluated using 3H thymine incorporation. The human iVSMCs were seeded at 3500 cells/0.32 cm2 in collagen 1 coated 60 mm dishes in SmGM-2 Smooth Muscle Growth Medium-2 (Cat #CC-3182, Lonza). After overnight culture, the cells were transfected with siRNA targets to human ENPP1 using Lipofectamine RNAiMAX (cat #13778500, ThermoFisher Scientific) for overnight. Following 48 hours starvation with 0.25% FBS, cells were stained with AOPI and counted with auto cell counter, Cellometer 2000. Cells were reseeded in well of 96 well plate at 2500 cells/well and cultured in complete medium in the presence and absence of Etidronate (Cat #P5248, Sigma) at indicated concentration. Cell proliferation was evaluated on day 3 by [3H] thymidine uptake. Results are expressed as CPM±SEM. Experiments were triplicated.

As shown in FIG. 24, bisphosphonate did not appear to inhibit proliferation in human iVSMCs.

Example 6: In Vivo Murine Carotid Artery Ligation Studies

Carotid artery ligation in the mouse is a common model for investigating the response of the vasculature to mechanical injury. Damage to the vessel induces an inflammatory response and endothelial activation, resulting in smooth muscle cell proliferation and narrowing of the lumen of the vessel. Accordingly, carotid artery ligation in the tip-toe-walking (ttw) mouse, which contains a mutation in Enpp1 and serves as a model of generalized arterial calcification of infancy (GACI), was used to investigate the role of Enpp1 treatment on intimal hyperplasia.

FIG. 25 shows the carotid ligation procedure. In anesthetized animals, the left carotid artery is exposed through a small incision in the neck and is ligated with a suture approximately 2 mm proximal from the carotid bifurcation. The animals are allowed to recover for 14 days, at which time the carotid arteries are harvested and fixed in 4% paraformaldehyde in PBS for sectioning and histological analysis. Serial sections of 5 μm were taken spanning 250 μm from the ligation. Every fifth section was analyzed with Von Gieson's stain and morphometric analysis was performed using Image J software.

To determine the effect of Enpp1 on intimal hyperplasia, 6 to 7-week old homozygous ttw/ttw mice were treated with either vehicle or recombinant human Enpp1 (rhEnpp1) at 10 mg/kg by subcutaneous (SC) injection every other day. The mice were treated for 7 days prior to carotid ligation, and treatment continued for 14 days post-surgery when the carotid arteries were harvested for analysis.

The histological analysis is shown in FIG. 26. Representative stained sections from either 100 μm (top) or 200 μm (bottom) from the ligation in WT and vehicle or rhEnpp1-treated ttw/ttw are shown from left to right, respectively. Von Gieson's solution stains elastic collagen fibers and distinguishes the internal (IEL) and external elastic lamina (EEL) from the lumen of the vessel (L). In the WT mice, the carotid ligation caused intimal hyperplasia resulting in narrowing of the lumen, with more severe narrowing closer to the ligature (100 μm) and less severe occlusion further away (200 μm). In contrast, in the ttw/ttw mice the degree of intimal hyperplasia appeared to be increased, as the lumen at 200 μm is almost completely occluded. The ttw/ttw mice treated with rhENPP1 showed much less intimal hyperplasia than those treated with vehicle, approaching the degree seen in WT animals. This suggests that the presence of Enpp1 prior to and after the carotid ligation protected against intimal hyperplasia.

FIGS. 27A-C show morphometric quantitation of the results. Measurement of the circumference of the external and internal elastic lamina and the luminal border allows quantitation of the medial (M) and intimal (I) areas. The medial area, between the external and internal lamina, remained constant (FIG. 27A). The intimal area within the lumen showed a statistically-significant increase in ttw/ttw and vehicle-treated ttw/ttw mice relative to WT mice (FIG. 27B). The rhENPP1-treated ttw/ttw mice were similar to WT mice in both the intimal area and the I/M ratio, with the results again being statistically significant (FIG. 27C). These results support the protective effect of rhENPPP1 when administered prior to carotid ligation.

In order to determine if ENPP1 could have a therapeutic effect if given after the carotid ligation, 6 to 7-week old ttw/ttw mice were subjected to carotid ligation and allowed to recover. rhEnpp1 treatment (10 mg/kg SC every other day) was initiated 7 days following carotid ligation and continued until the carotid arteries were harvested at 14 days post-ligation.

FIG. 28A shows the degree of intimal hyperplasia present at 100 and 200 μm 7 days post carotid ligation, prior to the initiation of ENPP1 treatment. Histological assessment of the therapeutic effect of rhENPP1 when initiated at 7 days post ligation is shown in FIG. 28B, with representative sections at 100 μm (top) and 200 μm (bottom) from the ligation in vehicle-treated (left) and rhENPP1-treated (right) ttw/ttw mice presented. Despite the beginning of some intimal hyperplasia in the untreated animals at 7 days post ligation (FIG. 28A), treatment with rhENPP1 beginning at this point still showed benefit as the degree of luminal occlusion at both 100 and 200 μm was less than in the vehicle-treated animals 14 days post ligation.

FIG. 29 (A-C) shows the morphometric quantitation of the data. The medial area, between the external and internal lamina, remained constant. The vehicle-treated ttw/ttw mice and rhENPP1-treated ttw/ttw mice had similar intimal area, both showed significant more proliferated intimal area than WT mice (p<0.01 and p<0.05, respectively). The I/M ratio of vehicle-treated ttw/ttw mice was increased compared to WT mice, with the results again being statistically significant. However, the I/MV ratio of rhENPP1-treated ttw/ttw mice was between the levels of WT and vehicle-treated ttw/ttw mice, not significantly different compared to WT and vehicle-treated ttw/ttw mice, indicating a decelerating effect of rhENPP1 on already started intimal proliferation.

In summary, in response to carotid artery ligation for two weeks, vehicle treated ttw/ttw mice showed accelerated neointimal hyperplasia. In contrast, ENPP1-Fc treated carotid ligated ttw/ttw mice displayed a significant reduction in intimal proliferation, comparable to the proliferation level of ligated WT mice. The results demonstrate that subcutaneous administration of recombinant ENPP1-Fc fusion protein prevents intimal hyperplasia in an animal model of GAC. This finding suggests that ENPP1 enzyme replacement is a potential therapeutic approach for treating intimal hyperplasia in GACI patients.

SUMMARY OF SEQUENCE LISTING SEQ ID NO: 1 amino acid sequence of wild-type NPP1 protein MERDGCAGGGSRGGEGGRAPREGPAGNGRDRGRSHAAEAPGDPQAAASLLAPMDVGE EPLEKAARARTAKDPNTYKVLSLVLSVCVLTTILGCIFGLK PSCAKEVKSCKGRCFERTF GNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCACSDDCKDKGD CCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVI SKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKE KFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVL QWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNL HRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYE GIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGS GFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSL NHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIK HETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLY QDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWR YFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVL TSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHI TGLSFYQQRKEPVSDILKLKTHLPTFSQED SEQ ID NO: 2 amino acid sequence of sNPP1 that contains cysteine-rich region, catalytic region and c-terminal region PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEK RLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSL DGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDN KMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDI YKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQR VDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGP AARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFY LDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYN LMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNP SILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPL WTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSG IYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSL ENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGK HDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED SEQ ID NO: 3 amino acid sequence of sNPP1-Fc fusion protein PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEK RLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSL DGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDN KMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDI YKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQR VDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGP AARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFY LDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYN LMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNP SILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPL WTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSG IYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSL ENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGK HDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4 amino acid sequence of sNPP1-Fc-D10 PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEK RLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSL DGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDN KMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDI YKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQR VDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGP AARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFY LDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYN LMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNP SILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPL WTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSG IYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSL ENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGK HDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK DDDDDDDDDD SEQ ID NO: 5 amino acid sequences of soluble NPP1 containing amino acids from 107 to 925 of SEQ ID NO: 1 SCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCA CSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYL HTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKM NASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGS VPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGML MDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDV PDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLAL NPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTP APNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQ FNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRND SFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIV PMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIR NQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELL MLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED SEQ ID NO: 6 amino acid sequence of soluble NPP1 containing amino acids from 187 to 925 of SEQ ID NO: 1 EKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTK NMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPI WVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERP HFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDH GMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREP NQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFS NMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVY TPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRP RVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSP VHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLR KYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTS QTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQ QRKEPVSDILKLKTHLPTFSQED SEQ ID NO: 7 amino acid sequence of Fc region of human IgG1 including hinge region EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 8 amino acid sequence of Fc of human IgG1 including partial hinge region DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 9 amino acid sequence of NPP1-Fc fusion protein [(107-925)-Fc] SCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCA CSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYL HTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKM NASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGS VPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGML MDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDV PDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLAL NPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTP APNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQ FNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRND SFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIV PMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIR NQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELL MLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10 amino acid sequence of NPPl-Fc fusion protein [(107-925)-partial hinge Fc] SCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCA CSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYL HTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKM NASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGS VPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGML MDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDV PDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLAL NPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTP APNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQ FNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRND SFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIV PMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIR NQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELL MLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 11 amino acid sequence of NPP1-Fc fusion protein [(187-925)-Fc] EKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTK NMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPI WVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERP HFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDH GMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREP NQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFS NMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVY TPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRP RVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSP VHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLR KYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTS QTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQ QRKEPVSDILKLKTHLPTFSQEDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO: 12 amino acid sequence of NPP1-Fc fusion protein [(187-925)-partial hinge Fc] EKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTK NMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPI WVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERP HFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDH GMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREP NQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFS NMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVY TPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRP RVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSP VHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLR KYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTS QTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQ QRKEPVSDILKLKTHLPTFSQEDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK SEQ ID NO: 13 human IgG1 hinge region EPKSCDKTHTCPPCP SEQ ID NO: 14 portion of human IgG1 hinge region DKTHTCPPCP SEQ ID NO: 15 portion of human IgG1 hinge region PKSCDKTHTCPPCP SEQ ID NO: 16 Linker (Gly₄Ser)₃ SEQ ID NO: 17 amino acid motif that is start of soluble NPP1 which includes cysteine rich region PSCAKE SEQ ID NO: 18 D10 targeting moiety 19 synthetic linker (Gly₄Ser)_(n), 

1.-23. (canceled)
 24. A method for determining the effect of ectonucleotide pyrophosphatase pyrophosphorylase 1 (NPP1) treatment on intimal hyperplasia in response to a mechanical injury to a vasculature in an animal model of generalized arterial calcification of infancy (GACI), wherein the animal model is a tip-toe walking (ttw) mouse, the method comprising: measuring intimal hyperplasia in the vasculature of the animal following treatment with an NPP1 polypeptide, wherein the animal has been administered the NPP1 polypeptide prior to mechanical injury, following mechanical injury, or both prior to and following a mechanical injury to the vasculature of the animal, wherein the mechanical injury is carotid artery ligation and comparing the intimal hyperplasia in the animal treated with the NPP1 polypeptide with a control animal that has been treated with vehicle and not with the NPP1 polypeptide, thereby determining the effect of treatment with the NPP1 polypeptide on intimal hyperplasia in response to the mechanical injury of the vasculature.
 25. The method of claim 24, wherein the NPP1 polypeptide has been administered subcutaneously.
 26. The method of claim 24, wherein the NPP1 polypeptide is a recombinant human NPP1 polypeptide or a recombinant NPP1 fusion protein comprising an Fc region of an immunoglobulin.
 27. The method of claim 24, wherein the intimal hyperplasia results in narrowing of the lumen of a vessel.
 28. The method of claim 24, wherein the NPP1 polypeptide has been administered prior to carotid artery ligation.
 29. The method of claim 24, wherein the NPP1 polypeptide has been administered prior to and following carotid artery ligation.
 30. A method for determining the effect of NPP1 treatment on intimal hyperplasia in response to a mechanical injury to a vasculature in an animal model of GACI, the method comprising: measuring intimal hyperplasia in the vasculature of the animal following treatment with an NPP1 polypeptide, wherein the animal has been administered the NPP1 polypeptide prior to mechanical injury, following mechanical injury, or both prior to and following a mechanical injury to the vasculature of the animal, wherein the NPP1 polypeptide is a recombinant human NPP1 polypeptide or a recombinant NPP1 fusion protein comprising an Fc region of an immunoglobulin, and wherein the mechanical injury is carotid artery ligation and comparing the intimal hyperplasia in the animal treated with the NPP1 polypeptide with a control animal that has been treated with vehicle and not with the NPP1 polypeptide, thereby determining the effect of treatment with the NPP1 polypeptide on intimal hyperplasia in response to the mechanical injury of the vasculature.
 31. The method of claim 30, wherein the NPP1 polypeptide is administered subcutaneously.
 32. The method of claim 30, wherein the intimal hyperplasia results in narrowing of the lumen of a vessel.
 33. The method of claim 30, wherein the NPP1 polypeptide has been administered prior to carotid artery ligation.
 34. The method of claim 30, wherein the NPP1 polypeptide has been administered prior to and following carotid artery ligation.
 35. The method of claim 34, wherein the animal model of GACI is a tip-toe-walking (ttw) mouse. 